All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Apoptosis is an active mechanism of cell death controlling the development and homeostasis of multicellular organisms. Tight regulation is required to ensure a delicate balance of life and death. Indeed, loss of apoptosis regulation results in a wide variety of diseases. Excess apoptosis might result in neurodegenerative disorders [Mattson, M. P. (2000) Nat. Rev. Mol. Cell Biol., 1: 120-129], reperfusion injury after ischemic episodes [Rathmell, J. C. and Thompson, C. B. (2002) Cell, 109 Suppl: S97-107], and immunodeficiency [Reed, C. J. (2000) Semin. Hematol., 37: 9-16]. On the other hand, lack of apoptosis is involved in cancer [LaCasse, E. C. et al. (1998) Oncogene, 17: 3247-3269], and autoimmune disorders [Rathmell, J. C. and Thompson, C. B. (2002) id ibid]. Several gene families are involved in the negative regulation of apoptosis, including the Inhibitor of Apoptosis Proteins (IAP). The products of the IAP gene family, discovered during the last five years, play a key role in apoptosis regulation and have become increasingly prominent in the field of cancer [Salvesen, G. S. and Duckett, C. S. (2002) Nat. Rev. Mol. Cell Biol., 3:401-410]. So far, eight human IAPs have been identified: c-IAP1, c-IAP2, NAIP, Survivin, XIAP, Bruce, ILP-2, and Livin. These proteins contain one or more repeats of a highly conserved 70 amino acids domain termed the baculovirus IAP repeat (BIR), located at the amino-terminal. With the exception of NIAP and Survivin, human IAPs contain a conserved sequence termed RING finger at the carboxy-terminal. IAPs can block apoptosis mainly through their ability to bind and inhibit specific caspases [Stennicke, H. R. et al. (2000) Biochem. J., 350 Pt 2: 563-568]. Initially, the molecular interaction between IAPs and caspases was thought to be mediated through the BIR domain [Takahashi, R. et al. (1998) J. Biol. Chem., 273: 7787-7790]. However, recent crystallographic resolution studies revealed that conserved amino acids in the linker region between BIR1 and BIR2 of XIAP are the most critical for its interaction with caspases 3 and 7. Surprisingly, the BIR2 domain itself has almost no direct contact with caspases 3 and 7 [Chai, J. et al. (2001) Cell, 104: 769-780; Huang, Y. et al. (2001) Cell, 104: 781-790; Riedl, S. J. et al. (2001) Cell, 104: 791-800]. The linker region preceding BIR2 can inhibit caspases through its ability to sterically hinder the substrate access. Yet, this region alone is not sufficient, and the BIR domain is required to either align or stabilize the structure. The BIR domain has also a regulatory function, as molecules such as SMAC/Diablo and HtrA2, that inhibit IAPs function, bind to this region [Hegde, R. (2002) J. Biol. Chem., 277: 432-438]. Several reports showed that many proteins containing a RING domain have E3-ubiquitin ligase activity. This activity is important in mediating the transfer of ubiquitin both to heterologous substrates as well as to the protein itself, thus targeting them to intracellular degradation [Suzuki, Y. et al. (2001) J. Biol. Chem., 276: 27058-27063; Yang, Y. et al. (2000) Science, 288: 874-877]. Indeed, several IAPs were shown to mediate RING-dependent ubiquitylation of caspases as well as to themselves [Jesenberger, V. (2002) Nat. Rev. Mol. Cell Biol., 3: 112-121]. Yet, the full potential of this function in apoptosis regulation remains unclear.
The essential role that IAPs play in the apoptotic process suggests that their activity must be tightly regulated. Indeed, it was reported that IAPs are regulated at the transcriptional/posttranscriptional levels as well as by interaction with inhibitory proteins [Vucic, D. et al. (2002) J. Biol. Chem., 277: 12275-12279]. Another important mechanism to negatively regulate IAPs is the ability of certain caspases, such as caspases 3 and 7 to specifically cleave these anti-apoptotic proteins [Ikeda, H. et al. (1997) Immunity, 6: 199-208].
Among the IAP family, XIAP and cIAP1 were shown to undergo a site-specific cleavage that is mediated by caspases [Deveraux, Q. L. et al. (1999) Embo J., 18: 5242-5251].
The present inventors and other groups have reported a novel IAP member, which was designated Livin/ML-IAP/KIAP [Vucic, D. et al. (2000) Curr. Biol., 10: 1359-1366; Lin, J. H. et al. (2000) Biochem. Biophys. Res. Commun., 279: 820-831; Kasof, G. M. (2001) J. Biol. Chem., 276: 3238-3246; Ashhab, Y. et al. (2001) FEBS Lett., 495: 56-60]. Livin contains a single BIR domain at the N-terminus and a carboxy-terminal RING domain. The inventors have previously demonstrated that Livin encodes two splicing variants, termed Livin α and β [Ashhab, Y. et al. (2001) id ibid]. These two proteins are highly similar, except for 18 amino acids located between the BIR and the RING domains, which are present in the α but not in the β isoform.
In the present invention, the inventors demonstrate that Livin undergoes site-specific cleavage by effector caspases 3 and 7, producing a large C-terminal subunit containing both the BIR and RING domains. Moreover, both Livin α and β undergo this cleavage, thus generating Livin-derived peptides α and β.
Unexpectedly, the inventors have found that the two Livin-derived peptides have pro-apoptotic properties. The present invention provides said pro-apoptotic peptides, together with compositions and uses thereof In addition, the present invention provides methods utilizing these Livin-derived peptides.
These and other objects of the present invention will become apparent as the description proceeds.